Process for the determination of peroxides

ABSTRACT

A process for semiquantitative colorimetric detection of organic peroxides, especially ether peroxides, with the aid of peroxide indicating means comprising an absorbent carrier impregnated with peroxides, a colorimetric indicator, optionally a buffer, and optionally other additives. The indicating means is immersed in a solution of peroxides in a volatile organic solvent having a boiling point of below 80* C., preferably of below 50* C., and withdrawn. The indicating means is placed in a water vapor atmosphere having a relatively constant temperature between about 20 and 100* C. to evaporate the organic solvent and deposit moisture on the surface thereof, and then examined for color change.

Schmitt et al.

Nov. 6, 1973 PROCESS FOR THE DETERMINATION OF PEROXIDES lnventors: Dieter Schmitt; Alfred Stein;

Wilhelm Biiumer, all of Darmstadt, Germany Assignee: Merck Patent Gesellschaft mit beschrankter Haftung, Darmstadt, Germany Filed: Mar. 21, 1972 Appl. No.: 236,791

Foreign Application Priority Data Mar. 26, 1971 Germany P 21 14 638.3

US. Cl. 23/230 R, 23/230 B, l95/103.5 R Int. Cl. G01n 31/22 Field of Search 23/230 R, 230 B;

References Cited UNITED STATES PATENTS 5/1965 Oakes 23/230 B X 2,595,755 7/1971 Hiirtcl l95/l03.5 R

Primary Examiner-Robert M. Reese Att0rneyl. William Millen et a1.

[57] ABSTRACT A process for semiquantitative colorimetric detection of organic peroxides, especially ether peroxides, with the aid of peroxide indicating means comprising an absorbent carrier impregnated with peroxides, a color-imetric indicator, optionally a buffer, and optionally other additives. The indicating means is immersed in a solution of peroxides in a volatile organic solvent having a boiling point of below 80 C., preferably of below 50 C., and withdrawn. The indicating means is placed in a water vapor atmosphere having a relatively constant temperature between about 20 and 100 C. to evaporate theorganic solvent and deposit moisture on the surface thereof, and then examined for color change.

10 Claims, N0 Drawings PROCESS FOR THE DETERMINATION OF PEROXIDES BACKGROUND OF THE INVENTION 1. Field Of The Invention 1 This invention relates to a colorimetric process for the semiquantitative detection of peroxides, especially ether peroxide, using a peroxidase-containing indicator.

2. DESCRIPTION OF THE PRIOR ART Semiquantitativedetection of peroxides, especially hydrogen peroxide, with the use of color-changing test strips is well-known in the art, and many suitable test strips are commerically available which indicate the presence of hydrogen peroxide by the color change of an indicator. The sensitivity of such test strps for organic peroxides, especially ether peroxides, generally is too low to permit serniquantitative determinations. Therefore, it has heretofore been possible .to attain only av low sensitivity qualitative indication of organic peroxides.

Conventional colorimetric indicating means used for the determination of hydrogen peroxide are e.g., test strips for detecting glucose in the urine, or indicating strips for the determination of H in foodstuffs. The indicating means normally comprise an absorbent carrier containing peroxidase, an oxidation-sensitive colori'metric indicator such as a chromogen, and optionally a buffer. The absorbent carrier is normally paper, preferably filter paper. Common colorimetric indicators for this purpose include but are not limited to otolidine, benzidine, o-dianisidine, and 2,7- diaminofluorene. As the buffer substances, alkali acetates or citrates are commonly employed, but borate, tartrate, succinate, orphosphate buffers are also suitable. Preferably, the test strips-also contain additional conventional additives, e.g. albumen, gelatin,'polyvinyl alcohol, and/or polyvinylpyrrolidone. The absorbent carrier is generally impregnated with the reagents, preferably by successive impregnation with two or more solutions. Usually, the first impregnating solution contains the colorimetric indicator dissolved in a volatile organic solvent. The carrier is next dried and then impregnated with an aqueous solution containing the buffer, the peroxidase, and optionally any further additives. The absorbent carrier is then generally glued or heat-sealed onto a metallic foil or plastic film strip to form the reaction zone proper of the thus-produced test strips.

The prior art technique for using these colorimetric test strips is well-known and involves immersion of the test strip in a solution to be tested for peroxide content, withdrawing the test strip, evaporating the solvent, wetting the teststrip with water to obtain a colorimetric reaction, and comparing any color produced with a standardized color scale.

gen peroxide, this method suffers several disadvantages in addition to its low sensitivity for organic peroxides,

' particularly ether peroxides. Reproducibility of results No rapid method has heretofore been available for accurately testing ether for the presence of ether peroxides. Conventional testing with iron (ll) salts and ammonium rhodanide is very cumbersome, while the use of potassium iodide starch paper is not sufficiently sensitive for most applications. Since both ether and ether peroxides are dangerous to handle, it would be highly desirable to have a quick and accurate method for testing ether for the presence of ether peroxide. The present invention fills such needs.

Accordingly, it is an object of this invention to provide an improved colorimetric process for the detection of peroxides.

Another object of this invention is to provide a method for developing colorimetric test strips used to detect organic peroxides.

A further object of this invention is to provide a process which improves the sensitivity of colorimetric peroxide test strips.

An additional object of this invention is to provide a semi-quantitative colorimetric method for the analysis of organic peroxides.

These and other objects of this'invention will become more fully apparent to those skilled in the art from the following description of the invention.

SUMMARY OF THE INVENTION Briefly, the present invention provides a process for the colorimetric detection of a peroxide which is soluble in a volatile organic solvent having a boiling point below about C., which comprises: (a) moistening peroxide indicating means comprising an absorbent carrier impregnated with peroxidase, at least one colorimetric redox indicator, and optionally a buffer, with a volatile organic solvent having a boiling point below about 80 C.; (b) removing said moistened indicating means from said volatile organic solvent and, without completely evaporating said solvent from said indicating means; (c) passing a saturating amount of moist gas having arelative humidity-of about 80 percent and a relatively constant temperature of between 20 100 C., onto said indicating means to evaporate said solvent from 'said indicating means and condense a water film onto the surface of said indicating means; and (d) observing'any resultant color change of said peroxide indicating means. In a particularly preferred embodiment, thisis achieved by forced breathing over the'colorimetric indicator as it is withdrawn from the organic solvent.

According to the present invention, the sensitivity of conventional colorimetric test strips can easily be increased sufficiently to conduct semiquantitative determinations of organic peroxides, especially ether peroxides. The sensitivity of conventional peroxide colorimetric indicating means can be raised by an order of magnitude so that even minor amounts of peroxides can be easily detected with excellent accuracy.

The indicating means is moistened, preferably by immersion in a solution of the peroxide in a readily volatile organic solvent having a boiling point of below 80 C., preferably of 10 to 50 C. According to the present invention, the peroxide indicating means is introduced into a water vapor atmosphere having a relatively constant temperature of about 20 100 C., preferably 36 100 C. before the volatile organic solvent has evaporated. The water vapor atmosphere preferably is at a temperature of at least 20 C. The use of a water vapor atmosphere having a temperature of about 36 C. and a relative humidity of about 90 percent is particularly advantageous with an ehter solvent and can be achieved in an extremely simple manner by forced breathing, preferably several times, over the reagent zone of the test strip while the organic solvent is still evaporating. Due to the evaporative cooling effect, exhaled moisture condenses as a water film on the test paper, and the indication reaction then takes place in this surface water film. Additionally, due to the evaporation of the organicsolvent, the peroxides which have been absorbed by the test paper during immersion are transported to the surface of the indicating means, where they are concentrated in the surface water film. In' this manner, the indicating means sensitivity is substantially increased. In comparison with a test strip which is conventionally moistened with sufficient water required for the indicating reaction after the organic solvent as evaporated, less than one tenth of the sensitivity achieved by this invention is attained, since the peroxides became distributed throughout the entire volume of the reaction zone.

While not wishing to be bound by any theory, it is believed that semiquantitative determination becomes possible because using a moist gas of constant temperature and humidity to saturate the reagent zone results in the formation of an almost exactly reproducible water film on the surface of this zone. In this method, the amount of the water condensing on the indicating reagent'surface is a function of the amount of absorbed and evaporating organic solvent as long as a sufficient amount of water vapor at a constant temperature is provided. The heat of condensation of the water vapor compensatesfor the evaporative cooling of the volatile.

organic solvent, and saturation of the reagnet zone with an amount of the moist gas sufficient to evaporate the solvent and deposit a water film on the reagent zone surface is effected. Since the amount of the absorbed and evaporated solvent is a direct function of the absorption capacity of the indicating means for this solvent, it is possible to' attain good reproducibility and calibration for a given type of test strip. A water vapor atmosphere of constant temperature and uniform moisture can be produced with surprising constancy by human breath which is fairly constant at 36 C. and 90 percent relative humidity, even under fluctuating ambient conditions, and this can be used to provide optimal conditions for a semiquantitative colorimetric determination of organic peroxides, especially ether peroxides. Of course, it is also possible to produce a suitable water vapor atmosphere having a temperature of between and 100 C., preferably 36 to 100 C. and a relative humidity of between 80 and 100 percent, preferably 85 to 95 percent in any other desired manner; for example, the test strip can be introduced into the water vapor rising from boiling water or .a water bath.

It is important to obtain reproducible results that a reproducible water film be deposited; this is best achieved when the relative humidity and temperature of the moist gas are constant.

Suitable volatile organic solvents are those having a boiling point of below 80 C., preferably below 50 C. Preferably, the volatile organic solvent is a peroxidefree ether, especially diethyl ether. Also suitable as an organic solvent are lower aliphatic hydrocarbons having this boiling range, especially petroleum ether; acetone; ethyl acetate; and methyl ethyl ketone. The only criticality is that the solvent selected possesses a sufficient latent heat of evaporation to provide condensation of a water film, generally from 4 to 10 kcal/moL, preferably from 5 to 8 kcal/mol.

Any organic peroxides which are soluble in the volatile organic solvents used can be detected by the process of this invention. in addition to detecting ether peroxides, the present procedure can also be used for the detection or titration of other organic peroxides, e.g. for diacyl peroxides, aldehyde peroxides, ketone peroxides, alkyl hydroperoxides, alkyl peresters and peracides (e.g. perbenzoic acid, performic acid, peracetic acid, perphthalic acid, permaleic acid). Routine laboratory determinations can be conducted for the determination of acyl and/or diacyl peroxides, e.g. dibenzoyl peroxide or lauroyl peroxide, cumene hydroperoxide (a, a-dimethylbenzyl hydroperoxide), methyl ethyl ketone hydroperoxide, and cyclohexanone hydroperoxide. This is surprising since it has not heretofore been appreciated that peroxidase is capable of transferring oxygen even from peroxides of poor water solubility.

The peroxides of higher ethers, which are not readily volatile, e.g. those boiling at 60 to 120 C., can likewise be easily detected according to the process of the present invention. According to this invention, the higherboiling ether to be examined is diluted with a peroxidefree, volatile ether, (e.g. diethyl ether) or with a volatile organic solvent; in this manner, precise semiquantitative peroxide determinations can be conducted.

In the process of this invention, all indicating means can be employed which are suitable for the colorimetric determination of hydrogen peroxide. However, the content of peroxidase in these indicating means should preferably be relatively high. The content of peroxidase in the impregnating solution used to prepare the indicating means is preferably at least 20 mg./ ml., especially about 30-50 mg./l00 ml.

The sensitivity of the process of this invention can also be further enhanced by increasing the thickness of the absorbent carrier portion of the reaction zone, since the peroxides absorbed by the volatile organic solvent are concentrated by the evaporation of the solvent at the outer surface of the indicator means which is exposedto air. Standardized color scales whereby peroxide concentration can be read off directly in p.p.m. can be prepared for each particular absorbent carrier material being used.

The process of this invention is preferably conducted by first immersing the test strip in the solution to be tested until the reaction zone is fully moistened. After withdrawal from the solution, the test strip is immediately (i.e. preferably within up to 4, in particular 0.5 1 seconds) breathed upon several times (e.g. three to four times) for several seconds, until the maximum color depth has been attained. The thus-produced coloring is then compared with a standard color scale. Additional breathing on the indicator means does not alter the color. The only function which must be achieved by breathing is complete wetting of the reaction zone with a water film; any excess water vapor does not have a disadvantageous effect. If the ether is allowed to evaporate before the reaction zone is moistened with water, only a very weak color change is obtained. Even evaporation of the organic solvent in normal, moist laboratory air, results in only a slight color change which is difficult or impossible to reproduce.

If, due to the greatly increased sensitivity of the process of this invention, a color is produced which is beyond the range of the standard color scale, dilution can be effected in the usual manner until the color produced is within the range of the standard color scale. By appropriate multiplication in accordance with the dilution used, the peroxide content can beaccurately determined.

The process of this invention is sufficiently senstiive to reliably detect 2 p.p.m. of ether peroxides. Using a suitable color scale, a marked distinction can be observed visually between concentrations of 5, 10, 30, 60, 100, 250, and 500 p.p.m. of ether peroxide, and even intermediate concentrations can be interpreted. Similar results are attained with color scales calibrated for other organic peroxides.

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosurein any way whatsoever.

EXAMPLE 1 Filter paper having a thickness of 0.35 mm. (Schleicher Schuell No. 1450 CV) is impregnated with a solution of l g. of o-tolidine in 100 ml. of methanol. After drying at 50i80 C., the paper is impregnated with a second solution containing, in 100 ml.'of water, 50mg. of-peroxidase, 5 g. of sodium citrate, and 1.5g. of albumen (pH 5). After drying, the papers are cut into 6 mm. strips and heat-sealed onto a plastic film backing. By cutting at right angles to the direction of the strips, test strips having a square reaction zone with a width of 6 mm. are prepared.

A test strip is immersed in a solution of 50 mg. of diethyl ether hydroperoxide in 1 liter of diethyl ether for about one second, so that the reaction zone is fully moistened. After withdrawal, the teststrip is immediately breathed upon 24 times for 3-5 seconds.

The thus-produced blue coloring is compared with a standardized color scale and indicates a peroxide content of 50 p.p.m. H

EXAMPLE 2 Test strips were prepared and tested as in Example 1,

using the following filter papers as absorbent carriers:

EXAMPLE 3 2 mg. of commercially obtained stabilized dibenzoyl peroxide to be tested for peroxide content is mixed with 100 ml. of peroxide-free diethyl ether. A test strip described in Example 1 is immersed therein and breathed upon after being withdrawn from the solution. Comparison with a standardized color scale showed a .color depth equivalent to an ether peroxide concentration of 5 mg./l. which, empirically determined, corresponds to a dibenzoyl peroxide content of mg./l.

This test indicated that the dibenzoyl peroxide sample had 50 percent strength.

Other acyl peroxides, e.g. lauroyl peroxide, as well as other hydroperoxides, e.g. cumene hydroperoxide, can be detected in the same manner.

Identical results are obtained using petroleum ether as a solvent (boiling point about 4045 C.) in place of ether. For frequent determinations of the same peroxide, the standardized color scale can be directly marked with these data.

The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the preceding examples.

From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

What is claimed is:

1. A process for the colorimetric detection of a pcroxide which is soluble in a volatile organic solvent having a boiling point below about C., which comprises:

a. moistening peroxide indicating means comprising an absorbent carrier impregnated with peroxidase, at least one colorimetric redox indicator, and optionally a buffer, with a volatile organic solvent having a boiling point below about 80 C.;

b. removing said moistened indicating means from said volatile organic solvent and, without completely evaporating said solvent from said indicating means;

c. within 4 seconds after said removing of said moistened indicating means from said volatile organic solvent, passing a saturating amount of moist gas having a relative humidity of about 80-100 percent and a relatively constant temprature of between 20-100 C., onto said indicating means to evaporate said solvent from said indicating means and condense a water film onto the surface of said indicating means; and

d. observing any resultant color change of said peroxide indicating means. I

2. A process according to claim 1, wherein said moist gas is air having a relative humidity of about per cent.

3. A process according to claim 1, wherein said moist gas is air having a temperature of about 36 C.

4. A process according to claim 1, wherein said moist gas is exhaledhuman breath.

5. A process according to claim 1, wherein said peroxide is a peroxide of a higher boiling ether, further comprising diluting said peroxide with a peroxide-free volatile organic solvent to form a mixture having a boiling poing of less than 80 C., and moistening said peroxide indicating means with said mixture.

6. A process according to claim 1, wherein said volatile organic solvent has a boiling point above about 36 C.

7. A process according to claim 1, wherein said peroxide is an ether peroxide.

' tile organic solvent is a peroxide-free ether.

9. A process according to claim 8, wherein said ether is diethyl ether.

10. A process according to claim 7, wherein said moist gas is exhaled human breath. 

2. A process according to claim 1, wherein said moist gas is air having a relative humidity of about 90 percent.
 3. A process according to claim 1, wherein said moist gas is air having a temperature of about 36* C.
 4. A process according to claim 1, wherein said moist gas is exhaled human breath.
 5. A process according to claim 1, wherein said peroxide is a peroxide of a higher boiling ether, further comprising diluting said peroxide with a peroxide-free volatile organic solvent to form a mixture having a boiling poing of less than 80* C., and moistening said peroxide indicating means with said mixture.
 6. A process according to claim 1, wherein said volatile organic solvent has a boiling point above about 36* C.
 7. A process according to claim 1, wherein said peroxide is an ether peroxide.
 8. A process according to claim 7, wherein said volatile organic solvent is a peroxide-free ether.
 9. A process according to claim 8, wherein said ether is diethyl ether.
 10. A process according to claim 7, wherein said moist gas is exhaled human breath. 